*Target (protein/gene name): D-Alanine Ligase *NCBI Gene # or RefSeq#: 985238 *Protein ID (NP or XP #) or Wolbachia#:NP_464381.1 *Organism (including strain):Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e) *Etiologic Risk Group:Risk group 2 *Disease Information: Listeriosis is a foodborne illness caused by infection with the bacterium Listeria monocytogenes. With a 21% case-fatality rate between 2009 and 2011 in the United States, listeriosis is the third highest cause of death of the major foodborne pathogens. Listeriosis is especially serious in unborn babies, newborn infants, pregnant women, elderly, and those with immunocompromised conditions and can result in fatal complications including miscarriages, septicemia, and meningitis. Listeria infection can result from eating unpasteurized milk products, raw produce, raw seafood, or improperly processed meat. Symptoms of listeriosis begin with fever, nausea, and diarrhea and progress to headaches, confusion, loss of balance, and convulsions. If symptoms persist and in high-risk patients, listeriosis is treated with ampicillin, often in combination with gentamicin, with the length of treatment varying by case *Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): https://www.patricbrc.org/portal/portal/patric/Feature?cId=PATRIC.169963.11.NC_003210.CDS.892662.893774.fwd&cType=feature http://www.ncbi.nlm.nih.gov/gene/985238 http://www.uniprot.org/uniprot/Q8Y8P1 *Essentiality of this protein: “D-Alanine ligase is involved in biosynthesis of peptidoglycan. In gram-positive bacteria, likely essential. Decrease of D-alanine ligase results in thinner cell walls and increased susceptibility to ampicillin when developing intracellularly.” http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900503/full *Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): predicted dimer *Complex of proteins?: No *Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019625/
Structure (PDB or Homology model) -- PDB for Homology model: 2I80 -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB:
---- Query Coverage: 99% coverage ---- Max % Identities: 48% identity ---- % Positives: 70% positive identity ---- Chain used for homology: A
Current Inhibitors: D-cycloserine Expression Information (has it been expressed in bacterial cells): http://www.ncbi.nlm.nih.gov/pubmed/1554356 Purification Method: Nickel affinity chromatography Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence: MKTKLILLYGGKSAEHEVSLQTAFSVINALDLEKFEAAPIYITNEGEWIQGPLLSGKLDF VEQLRFSATDTIKLATTESEKSEGEAISPAVLEADGQETVVFPLLHGPNGEDGTVQGLFE VLNIPYVGNGVLASSAAMDKIVMKKIFADAGIPQVPAVAVRLIDWKNYQEEMVAEMEEVL TYPVFVKPANLGSSVGISKATNKKELVDAMTEAFLYDRRVVVEQGVVAREIEMGVLGNDT PVCSVPGEILPEGAVATFYDYKAKYQDNNTALIIPTEVDPEILEQMKEYAIQAFLGLDAS GLVRADFFLTEDNQLFLNEVNTMPGFTPYSMYPLLWQETGLPYGALIERLVDLAKERHAA KNALKYKLED *Length of your protein in Amino Acids: 370aa Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 40.6695 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 37360 M-1 cm-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence: ATGAAAACTAAATTAATTTTATTATACGGTGGAAAATCTGCCGAACATGAAGTTTCATTACAAACAGCAT TTTCAGTTATCAATGCTTTGGATTTAGAAAAATTCGAAGCTGCGCCAATATATATTACGAATGAAGGTGA GTGGATTCAAGGACCACTTCTTTCTGGGAAGTTAGATTTTGTCGAACAATTACGTTTTTCGGCTACAGAT ACAATAAAACTTGCTACAACTGAATCAGAAAAATCAGAAGGGGAAGCAATTAGCCCGGCCGTTTTAGAAG CAGACGGACAAGAGACAGTCGTATTCCCACTTTTACATGGTCCAAACGGAGAAGATGGCACTGTTCAAGG ATTGTTCGAAGTATTAAACATTCCATATGTTGGCAACGGGGTCTTGGCATCTTCTGCTGCAATGGACAAA ATCGTGATGAAGAAAATTTTTGCAGATGCTGGAATCCCGCAAGTTCCAGCAGTTGCTGTTCGTTTAATTG ATTGGAAAAATTATCAAGAGGAAATGGTTGCTGAGATGGAAGAAGTCCTTACATATCCTGTCTTCGTAAA ACCAGCAAACCTTGGTTCGAGTGTTGGTATTAGTAAGGCAACAAACAAAAAAGAATTAGTGGACGCAATG ACAGAAGCGTTTTTATATGACCGTCGTGTTGTTGTTGAACAAGGTGTCGTTGCTCGTGAAATCGAAATGG GTGTACTTGGAAATGATACGCCAGTTTGCTCTGTACCTGGCGAAATTTTGCCAGAAGGTGCAGTTGCCAC ATTCTACGATTACAAAGCTAAATATCAAGATAACAATACTGCGTTAATTATTCCGACTGAAGTAGATCCA GAAATCTTGGAACAAATGAAAGAATATGCGATCCAAGCGTTTTTAGGTCTGGACGCAAGCGGACTAGTGC GAGCGGACTTTTTCTTAACGGAAGATAATCAGTTATTCTTAAATGAAGTGAATACAATGCCAGGTTTCAC ACCGTACAGCATGTATCCTCTTCTTTGGCAGGAAACGGGCTTACCGTACGGAGCGTTAATTGAACGATTA GTCGATTTAGCGAAAGAACGCCACGCAGCCAAAAATGCACTTAAATATAAATTAGAAGACTAA *GC% Content for gene:39.712%
Primer design results for 'tail' primers (this is just 2 sequences): Forward Primer: 5'-TACTTCCAATCCATGAAAACCAAACTGATTCTG-3' Reverse Primer: 5'-TATCCACCTTTACTGTTAGTCTTCGAGTTTGTATTTCA-3'
*NCBI Gene # or RefSeq#: 985238
*Protein ID (NP or XP #) or Wolbachia#: NP_464381.1
*Organism (including strain): Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
*Etiologic Risk Group:Risk group 2
*Disease Information:
Listeriosis is a foodborne illness caused by infection with the bacterium Listeria monocytogenes. With a 21% case-fatality rate between 2009 and 2011 in the United States, listeriosis is the third highest cause of death of the major foodborne pathogens. Listeriosis is especially serious in unborn babies, newborn infants, pregnant women, elderly, and those with immunocompromised conditions and can result in fatal complications including miscarriages, septicemia, and meningitis. Listeria infection can result from eating unpasteurized milk products, raw produce, raw seafood, or improperly processed meat. Symptoms of listeriosis begin with fever, nausea, and diarrhea and progress to headaches, confusion, loss of balance, and convulsions. If symptoms persist and in high-risk patients, listeriosis is treated with ampicillin, often in combination with gentamicin, with the length of treatment varying by case
*Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
https://www.patricbrc.org/portal/portal/patric/Feature?cId=PATRIC.169963.11.NC_003210.CDS.892662.893774.fwd&cType=feature
http://www.ncbi.nlm.nih.gov/gene/985238
http://www.uniprot.org/uniprot/Q8Y8P1
*Essentiality of this protein: “D-Alanine ligase is involved in biosynthesis of peptidoglycan. In gram-positive bacteria, likely essential. Decrease of D-alanine ligase results in thinner cell walls and increased susceptibility to ampicillin when developing intracellularly.” http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900503/full
*Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): predicted dimer
*Complex of proteins?: No
*Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019625/
*EC#: 6.3.2.4
*Link to BRENDA EC# page:http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.4
-- Show screenshot of BRENDA enzyme mechanism schematic
*Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Spectrophotometric assay - Malachite Green/BIOMOL Green
-- link to Sigma/company page for assay: http://www.enzolifesciences.com/BML-AK111/biomol-green/
-- -or link (or citation) to paper that contains assay information: http://www.pnas.org/content/early/2015/03/11/1418820112.full.pdf
-- links to assay reagents (substrates) pages:
http://www.enzolifesciences.com/BML-KI102/1ml-phosphate-standard-800-microm-for-biomol-green/
http://www.enzolifesciences.com/BML-AK111/biomol-green/
--- List cost and quantity of substrate reagents, supplier, and catalog #
BIOMOL Green, Enzo Life Sciences, Catalog No. BML-AK111-0250……..$123.00
(includes 250ml BIOMOL Green reagent and 800ul phosphate standard)
Structure (PDB or Homology model)
-- PDB for Homology model: 2I80
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB:
---- Query Coverage: 99% coverage
---- Max % Identities: 48% identity
---- % Positives: 70% positive identity
---- Chain used for homology: A
Current Inhibitors: D-cycloserine
Expression Information (has it been expressed in bacterial cells): http://www.ncbi.nlm.nih.gov/pubmed/1554356
Purification Method: Nickel affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence:
MKTKLILLYGGKSAEHEVSLQTAFSVINALDLEKFEAAPIYITNEGEWIQGPLLSGKLDF
VEQLRFSATDTIKLATTESEKSEGEAISPAVLEADGQETVVFPLLHGPNGEDGTVQGLFE
VLNIPYVGNGVLASSAAMDKIVMKKIFADAGIPQVPAVAVRLIDWKNYQEEMVAEMEEVL
TYPVFVKPANLGSSVGISKATNKKELVDAMTEAFLYDRRVVVEQGVVAREIEMGVLGNDT
PVCSVPGEILPEGAVATFYDYKAKYQDNNTALIIPTEVDPEILEQMKEYAIQAFLGLDAS
GLVRADFFLTEDNQLFLNEVNTMPGFTPYSMYPLLWQETGLPYGALIERLVDLAKERHAA
KNALKYKLED
*Length of your protein in Amino Acids: 370aa
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 40.6695 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 37360 M-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence:
ATGAAAACTAAATTAATTTTATTATACGGTGGAAAATCTGCCGAACATGAAGTTTCATTACAAACAGCAT
TTTCAGTTATCAATGCTTTGGATTTAGAAAAATTCGAAGCTGCGCCAATATATATTACGAATGAAGGTGA
GTGGATTCAAGGACCACTTCTTTCTGGGAAGTTAGATTTTGTCGAACAATTACGTTTTTCGGCTACAGAT
ACAATAAAACTTGCTACAACTGAATCAGAAAAATCAGAAGGGGAAGCAATTAGCCCGGCCGTTTTAGAAG
CAGACGGACAAGAGACAGTCGTATTCCCACTTTTACATGGTCCAAACGGAGAAGATGGCACTGTTCAAGG
ATTGTTCGAAGTATTAAACATTCCATATGTTGGCAACGGGGTCTTGGCATCTTCTGCTGCAATGGACAAA
ATCGTGATGAAGAAAATTTTTGCAGATGCTGGAATCCCGCAAGTTCCAGCAGTTGCTGTTCGTTTAATTG
ATTGGAAAAATTATCAAGAGGAAATGGTTGCTGAGATGGAAGAAGTCCTTACATATCCTGTCTTCGTAAA
ACCAGCAAACCTTGGTTCGAGTGTTGGTATTAGTAAGGCAACAAACAAAAAAGAATTAGTGGACGCAATG
ACAGAAGCGTTTTTATATGACCGTCGTGTTGTTGTTGAACAAGGTGTCGTTGCTCGTGAAATCGAAATGG
GTGTACTTGGAAATGATACGCCAGTTTGCTCTGTACCTGGCGAAATTTTGCCAGAAGGTGCAGTTGCCAC
ATTCTACGATTACAAAGCTAAATATCAAGATAACAATACTGCGTTAATTATTCCGACTGAAGTAGATCCA
GAAATCTTGGAACAAATGAAAGAATATGCGATCCAAGCGTTTTTAGGTCTGGACGCAAGCGGACTAGTGC
GAGCGGACTTTTTCTTAACGGAAGATAATCAGTTATTCTTAAATGAAGTGAATACAATGCCAGGTTTCAC
ACCGTACAGCATGTATCCTCTTCTTTGGCAGGAAACGGGCTTACCGTACGGAGCGTTAATTGAACGATTA
GTCGATTTAGCGAAAGAACGCCACGCAGCCAAAAATGCACTTAAATATAAATTAGAAGACTAA
*GC% Content for gene:39.712%
*CDS Gene Sequence (codon optimized):
ATGAAAACCAAACTGATTCTGCTGTATGGTGGTAAATCTGCGGAACACGAAGTTTCCCTGCAGACCGCGTTCTCTGTTATCAATGCACTGGACCTCGAAAAATTCGAAGCGGCTCCGATCTACATCACCAACGAGGGTGAATGGATTCAGGGTCCGCTCCTGTCTGGTAAACTGGACTTCGTTGAACAGCTCCGTTTCTCTGCGACCGATACCATCAAACTGGCGACCACCGAATCTGAAAAGTCTGAAGGTGAAGCGATCTCTCCGGCGGTTCTGGAAGCGGACGGTCAAGAAACCGTTGTTTTCCCGCTGCTGCACGGTCCGAACGGTGAAGACGGTACTGTGCAGGGTCTGTTCGAAGTTCTGAACATCCCGTACGTTGGTAACGGTGTTCTGGCGTCCTCTGCGGCGATGGACAAAATCGTTATGAAGAAAATCTTCGCGGACGCGGGTATTCCGCAAGTACCGGCCGTTGCGGTTCGTCTGATCGACTGGAAGAATTACCAAGAGGAGATGGTTGCAGAAATGGAAGAGGTTCTGACCTACCCGGTCTTCGTTAAACCGGCGAATCTCGGTTCTTCTGTTGGTATCTCTAAAGCGACCAACAAAAAAGAACTGGTCGATGCGATGACTGAAGCGTTCCTGTACGACCGTCGCGTTGTTGTTGAGCAAGGTGTGGTTGCCCGTGAAATCGAGATGGGCGTACTGGGTAACGACACCCCGGTTTGCTCTGTACCGGGTGAGATCCTGCCGGAAGGTGCAGTTGCGACCTTCTATGACTACAAAGCGAAATACCAGGACAATAACACCGCCCTGATCATCCCTACCGAGGTTGACCCGGAAATCCTGGAACAGATGAAAGAGTACGCGATCCAGGCCTTCCTGGGTCTGGACGCCTCTGGTCTCGTTCGTGCGGACTTCTTCCTCACCGAAGACAACCAGCTCTTCCTGAATGAAGTTAACACCATGCCGGGTTTCACCCCGTATTCTATGTACCCACTCCTGTGGCAGGAGACCGGCCTGCCGTATGGCGCTCTGATTGAACGTCTGGTTGATCTGGCGAAAGAACGTCACGCGGCGAAAAACGCGCTGAAATACAAACTCGAAGACTAA
*GC% Content for gene (codon optimized): 51%
Primer design results for pNIC-Bsa4 cloning:
DNA Works output
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer: 5'-TACTTCCAATCCATGAAAACCAAACTGATTCTG-3'
Reverse Primer: 5'-TATCCACCTTTACTGTTAGTCTTCGAGTTTGTATTTCA-3'